title: Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand.
abstract: To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.
authors.affiliation: Department of Physiology and Biophysics, University of Illinois, Urbana.
authors.forename: C K
authors.initials: CK
authors.lastname: Wrenn
authors.forename: B S
authors.initials: BS
authors.lastname: Katzenellenbogen
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics*; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives; D004965:Estrogen Antagonists / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D053284:Polyunsaturated Alkamides; D012319:RNA Polymerase II / Q000235:genetics; D011960:Receptors, Estrogen / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D012261:Ribonucleoproteins / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D004965:Estrogen Antagonists; D008024:Ligands; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen; D012261:Ribonucleoproteins; D005557:Formaldehyde; D004958:Estradiol; C051781:ICI 164384; D004247:DNA; D012319:RNA Polymerase II